This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Microcins are modified peptides that inhibit growth of competing Gram-negative bacteria such as Escherichia, Salmonella, and Enterobacter. Microcin C7 (MccC7) is produced by E. coli to eradicate competitors through a "Trojan horse" mechanism. After import into target cells, MccC7 is cleaved by nonspecific peptidases to release a toxic adenylated-aspartic-acid mimic that targets aspartyl-tRNA synthetase. MccC7 is generated by modification of a precursor heptapeptide, MccA (sequence MRTGNAN). Posttranslational steps in MccC7 biosynthesis involve conversion of the C-terminal Asn7 to an Asp amide with the nitrogen phosphoramidate-linked to AMP, followed by aminopropylation of a phosphate oxygen. Migration of the Asn7 carboxamido nitrogen and the N-P bond-forming step are catalyzed by the enzyme MccB. In a conventional acyl-adenylation reaction that consumes one ATP, MccB catalyzes adenylation of the MccA C-terminus. After an intramolecular rearrangement, an MccA peptidyl-succinimide intermediate is formed. Next, MccB catalyzes an unusual adenylation of the succinimide: the succinimidyl nitrogen attacks the [unreadable]-phosphate of a second ATP molecule, linking AMP to the peptide terminus via an N-P bond. The succinimide ring is hydrolyzed by regiospecific water-mediated opening to yield to the peptidyl-acyl-N-P-Adenosine group that is the Trojan horse reagent. MccB's N-terminal ~90 residue region is not detectably homologous to known structures. MccB's C-terminal ~260 residues share homology with a portion of ubiquitin-like protein (UBL) activating enzymes, also called E1s, which initiate UBL conjugation. This sequence similarity raises several questions about common and divergent aspects of MccB and E1 mechanisms. First, how does MccB recognize a heptapeptide substrate, whereas other family members have a e8 kDa UBL substrate? Second, why does MccB catalyze adenylation of a peptide C-terminal Asn or succinimide, rather than the Gly-Gly sequence found at UBL C-termini? Third, how can MccB catalyze two successive adenylation reactions? To address these questions, we analyzed MccB-MccA-nucleotide interactions.